Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Fusion of cells
Reexamination Certificate
1999-06-08
2001-04-24
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Fusion of cells
C435S285200
Reexamination Certificate
active
06221665
ABSTRACT:
TECHNICAL FIELD
The present invention relates to an electrofusion chamber, and particularly to a disposable electrofusion chamber which is used to provide a simple, inexpensive and efficient way of performing electrofusion.
BACKGROUND
Electrofusion is the common name for procedures that induce fusion of living cells using electricity. Cell-cell electrofusion (CCE) is the generic term used to describe electrofusion of living cells. CCE can refer to fusion of one cell type to a different cell type, or it can refer to fusing cells of the same type. In this application, reference to fusion of cells is intended to encompass both fusion of different cell types and also fusion of the same cell types. Moreover, it is intended to encompass the fusion of two or more cells to each other.
CCE processes generally involve three principal steps. First, fusion partners (i.e., two or more cells to be fused to each other) must be forced into contact with each other between two electrodes or some other means of inducing electrofusion. The cells must be in an electrically conductive medium. Second, one or more electrical pulses are applied to the cells that are in contact between the electrodes. Electrical pulses induce fusion and are administered by creating and maintaining a potential (voltage) difference across the electrodes. CCE is usually achieved using direct current (DC) pulses. The third and final CCE step occurs naturally; fused cells anneal into one cell due to their normal fluidity. CCE processes do not normally yield 100% fusion. Typically, a fraction of the contacted cells are induced to fuse while the remaining fraction does not fuse. Also, many of the extensively used methods involve steps which have a high rate of cell killing.
Most existing commercial CCE devices and applications known to the applicants use a process called dielectrophoresis to cause cell-cell contact. Dielectrophoresis is the application of alternating current (AC) to cause fusion partners to line up in chains between the electrodes. Thus, cell-cell contact is achieved at the points where adjacent cells in a chain are touching. Dielectrophoresis is incorporated into the first step of the three-step fusion process described above. After chains have formed, one or more DC pulses are delivered to induce fusion and the cells are allowed to anneal.
Jaroszeski et al., (Biophysical Journal, Vol. 67, Oct. 1994, 1574-1581) discloses apparatus and methods developed to enable mechanically facilitated cell-cell electrofusion to be performed. The apparatus and methods mechanically place cells in contact before fusion. A novel fusion chamber is disclosed composed of two functionally identical electrodes that are housed in a multi-layer structure. The layers function as a support for the electrodes. They also allow adjustment of the distance between opposing electrode faces. The electrodes were constructed to allow cells to be deposited, by vacuum, onto each face. The electrode faces were positioned at a predetermined distance from each other to mechanically force cell-cell contact between the deposited cells. Fusion was induced by delivering direct current pulses to the juxtaposed cells.
Jaroszeski et al. (Analytical Biochemistry, 216, 271-275 (1994)) discloses a cytometric method for detecting and quantitating hybrid cells that resulted from cell-cell electrofusion. Cells from two different lines and two vital fluorescent dyes were used in conjunction with a flow cytometer to demonstrate the method.
The German Patent Publication DE 3505147 A1 to Strellrecht et al. discloses an electrofusion process wherein cells are fixed on a first and second carrier. The two carriers are arranged so that the cells that are fixed to the respective carriers are opposite relative to each other. The cells are moved toward each other forming pairs of cells, one from each carrier. The pairs are each fused.
It would be advantageous to provide more efficient and effective means for inducing cell-to-cell contact and fusion than that described above. The present invention provides improved means for inducing such cell-cell contact and uses electric pulses applied from a different direction relative to deposited cells than prior art.
SUMMARY OF THE PRESENT INVENTION
The present invention provides a new and useful electrofusion device which is designed to be a convenient, inexpensive, and easy-to-use device that can be used to force cell-cell contact and to induce fusion of at least a portion of the cells in cell-to-cell contact. This device can be produced as a single and/or multiple use device, it is easily sterilized, it can be made as a disposable device, and does not require the use of AC.
Fusion without AC has significant benefits. For example, an electrofusion device that requires only a DC generator represents a lower initial equipment investment than is generally required for dielectrophoresis equipment. Moreover, the high cost of generators that produce AC and DC may discourage some researchers from using electrofusion. The present invention solves that problem by requiring only DC voltage, thereby enabling performance of electrofusion without the AC generator costs associated with conventional dielectrophoresis devices.
Additionally, the device of the present invention is flexible, in the sense that it can be operated from the DC power supply of various electric cell manipulator devices. For example, many laboratories use a physical phenomenon that is related to fusion in order to manipulate cells. This phenomenon is called electroporation. Specifically, it is common for researchers to perform both electrofusion and electroporation. However, electric pulse generators for electroporation produce DC pulses only. Thus, an inexpensive electrofusion chamber that does not require AC makes electrofusion possible for facilities that already have DC pulse generators.
The fact that in both of the foregoing examples the need for AC is eliminated also has biological relevance. Specifically, other devices known to applicants utilize a phenomenon called dielectrophoresis induced by AC of relatively long duration (e.g. seconds and/or minutes) to achieve cell-cell contact. Elimination of long duration AC is biologically advantageous because it can directly cause cellular damage. Also, heat generated during dielectrophoresis can be damaging to the cells.
According to the present invention, an apparatus for producing electrofusion of two types of cells comprises:
a. chamber with a substrate that is used as a surface for achieving cell-cell contact,
b. a mechanism for directing cells to be fused contained in a fluid medium toward one side of the substrate in such a manner that a substantial amount of cells are drawn to and retained against the one side of the substrate with a portion of the cells in cell-to-cell contact with each other along the one side of the substrate, and
c. a device for inducing fusion of a portion of the cells in cell-to-cell contact with each other over the predetermined portion of the one side of the substrate.
The device for inducing fusion preferably comprises a DC voltage, which is preferably disposed to apply an electric field to the substrate in an orientation which is parallel to the one side of the substrate. The DC voltage source is preferably applied to a pair of electrodes spaced apart along a predetermined portion of the side of the substrate, to apply DC voltage to the cells in cell-to-cell contact between the pair of electrodes.
The means for drawing the cells toward one side of the substrate can be achieved in two preferable ways that both achieve the same result. The first employs a porous substrate and a vacuum source. The vacuum source is configured to apply a level of vacuum to the fluid medium that is sufficient to draw a significant portion of the fluid medium through the substrate while retaining the cells on one side of the substrate in cell-to-cell contact. The vacuum source is configured to induce this migration of the cells and also to retain enough medium mixed with the cells to preserve the viability of th
Gilbert Richard A.
Heller Richard
Jaroszeski Mark J.
Ketter James
Kohn & Associates
University of South Florida
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